Preparation of specific antibody against CML, one of major AGE structures

Abstract

Nε-(Carboxymethyl)lysine (CML) is one of the major structures of advanced glycation endproducts (AGE). Immunological studies using monoclonal (6D12) and polyclonal anti-CML antibody demonstrated CML-accumulation in several pathological tissues such as kidneys of patients with diabetic nephropathy and atherosclerotic lesions of arterial walls. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly by glycolysis and polyol pathways, reacts with proteins to form MG-derived AGE structures such as Nε-(carboxyethyl)lysine (CEL), imidazolone and imidazolium salt cross-link like methylglyoxal-lysine dimmer (MOLD). Our preliminary studies demonstrated that 6D12 and polyclonal anti-CML antibodies significantly reacted not only with CML but also with CEL. In the present study, we determined the possibility that antibody could recognize a difference between CML and CEL. To prepare polyclonal CML-specific antibody (PCMS), rabbits were immunized with CML-keyhole limpet hemocyanin (CML-KLH) and CEL-reactive antibody was removed by a CEL-conjugated affinity chromatography. Similar to PCMS, polyclonal CEL-specific antibody (PCES) was purified by separating CML-reactive antibody from antiserum obtained by immunization with CEL-KLH. Next, the monoclonal antibody specific for CML (CMS-10) was obtained by immunization of CML-KLH, followed by successive screening by CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both PCMS and CMS-10 showed a significant reactivity to CML-proteins but not to CEL-proteins. In sharp contrast, PCES showed a high reactivity toward CEL-protein, but not to CML-protein. It is likely therefore that these antibodies were able to recognize the difference in one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with glucose-modified BSA (AGE-BSA) as well as BSA modified by several aldehydes such as glyoxal and 3-deoxyglucosone, potential intermediates for AGE formation. These results indicate that CMS-10 would help detect CML-modified proteins in vitro in a more specific way.